The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
6-Mercaptopurine (6-MP) or its pro-drug azathioprine (Imuran) is an immune system suppressant that is widely used to treat Inflammatory Bowel Disease (IBD), Ulcerative Colitis (UC), Crohn's Disease, Lupus, and in some cases, Rheumatoid Arthritis. In addition, Imuran is widely used to minimize organ rejection following transplants, especially of the kidneys. In 1953, 6-MP was introduced as a novel medication to induce remission in children with Acute Lymphocytic Leukemia (ALL). Prior to the introduction of 6-MP, ALL had a 100% fatality rate. Less commonly, a closely related purine, 6-thioguarine, is used instead of 6-MP.
Thiopurine-S-methyltransferase, TPMT enzyme, is a key enzyme in the pathway which converts 6-MP to the active nucleotide form. The gene for TPMT is polymorphic in all populations and gives the classic tri-modal distribution of TPMT activity. About 1 in 300-500 patients will be homozygous for a mutation which leads to rapid proteolytic degradation of TPMT. Thus, there may be homozygous, heterozygous, and wild-type individuals in a population. The approximate distribution is 0.3%, 11%, and 89%, respectively, in a random sampling of the general public. For homozygous patients, where TPMT enzyme has nominally zero activity, extraordinary high levels of the bioactive nucleotides (6-thioguaninenucleotides) are produced. In such individuals, excessive, and potentially lethal, immunosuppression can occur. These patients cannot receive 6-MP therapy safely at any dosage and it is important to identify them prior to initiating 6-MP therapy. Heterozygous individuals comprise about 9-11% of the population. For these patients, the physician typically reduces the dosage of 6-MP by about 50%.
A very small number of wild-type patients will have very high levels of TPMT. This is potentially problematic because early in the metabolic pathway, TPMT acts on the substrate 6-MP to form the potentially toxic metabolic product 6-methylmercaptopurine (6-MMP). Thus, an individual with very low TPMT produces excessively high levels of bioactive nucleotides, whereas a patient with very high TPMT produces excessive 6-MMP and may be at risk for hepatoxicity. To achieve optimal patient care, it may be desirable to know a patient's level of activity of TPMT enzyme prior to initiating Imuran therapy.
Various methods for measuring TPMT methylation products in a sample have been reported. Various methods have further reported use of these methods to determine TPMT enzyme activity in a sample. For example, Weinshilboum, R., et al., Clinica Chemica Acta, 1979, 97:59-71 reports using a HPLC-radioassay to detect TPMT enzyme products and to determine enzyme activity; Wan, W., et al., Rapid Comm. Mass Spectrom. 2004, 18:319-24, reports using MALDI-TOF mass spectrometry to detect TPMT generated 6-MMP and isotopically labeled 6-MMP in bacterial cell lysates; Kalsi, K., et al., Nucleosides, Nucleotides, and Nucleic Acids 2006, 25:1241-4, reports measuring TPMT enzyme activity in isolated red blood cells through detection of TPMT generated 6-MMP with ion trap mass spectrometry; Anglicheau, D., et al., J. Chromatogr. B. 2002, 773:119-27, reports utilization of HPLC with UV detection of 6-MMP to determine TPMT enzyme activity from isolated red blood cells; Breen, D., et al., Liver Transplantation 2005, 11:826-33, reports utilization of tandem mass spectrometry to detect TPMT generated 6-MMP from isolated red blood cells; and O'Brian, et al., U.S. patent application Ser. No. 11/370,581 (filed Mar. 8, 2006), reports utilization of tandem mass spectrometry to detect TPMT generated 6-MMP from isolated red blood cells through monitoring the reaction of a precursor ion at a mass to charge ratio of 165 to a fragment ion at a mass to charge ratio of 150.